Project Summary/Abstract This proposal addresses a novel transcriptional mechanism of antiviral resistance to major human pathogenic viruses. We found that pharmacological suppression of bromodomain-containing BET proteins (BRD2, 3 and 4) by small-molecular weight BET inhibitor (I-BET) result in increased viral replication of human viruses such as Influenza A, Yellow Fever and Dengue Fever viruses in human cells. Most strikingly, the treatment of mice with I-BET in vivo enables the Dengue Fever virus to replicate in the non-permissive host. In extension of these studies, we found that much of the pro-viral effect of BET inhibition is likely to be attributed to the suppression of BRD3. The siRNA-mediated inactivation of individual BET proteins in cultured human cells shows the selective and highly potent role of BRD3, but not BRD4 or BRD2, in the suppression of virus replication in vitro. Our search for the cause of selective BRD3 involvement in antiviral response revealed a novel mechanism that may contribute to the selective targeting of BRD3 to type I interferon (IFN) stimulated genes (ISG), We found that viral infection or treatment of human cultured cells with IFN leads to BRD3 association with transcription factor IRF9, that plays an important role in ISG expression. We found that association with IRF9 depends on the C-terminal serine-rich micro-domain in BRD3 that becomes phosphorylated in IFN treated cells. Substitution of serine 443 (Ser443) that has been identified as the most abundant site for the BRD3 phosphorylation, abolishes interaction between BRD3 and IRF9 in the IFN-treated A549 cells. Collectively, our data suggest the mechanism where IFN-triggered phosphorylation of BRD3 contributes to its binding to IRF9 followed by BRD3 recruitment to ISGs and activation of the ISG expression. In this proposal, we will address the role of BRD3 phosphorylation in BRD3 targeting to antiviral gene loci and regulation of antiviral gene transcription. Using CRISPR-Cas9-mediated mutagenesis, we will test the functional significance of the BRD3-controlled RNAs, including enhancer and non-coding RNA, in regulation of viral infection. We will also test whether overexpression of the individual BRD3-controlled RNAs in BRD3- deficient cells can restore the impaired antiviral response. Collectively, our studies will describe a novel mechanism of the antiviral gene regulation and provide information about protein-coding as well as non-coding RNA that may play a key role in antiviral response. This information could be highly valuable for the development of novel antiviral therapeutic approaches as well as for the control of inflammation associated with increased production of pro-inflammatory antiviral mediators. This aspect of our work has significant health relevance, since it deals with the possibility of reducing the severity of virus-associated hyper- inflammatory syndromes.